The use of vaccines is an effective method for disease control. However, preventive effects obtained by such methods are not sufficient in some cases. In this case, there are various possible reasons which include originally weak immunogenicity of an antigen designed as a vaccine or immunity of an individual to be vaccinated such as a newborn or an elderly person. ST, a causative toxin of coliform diarrhea, is a small molecule comprising 18 amino acids (STp (porcine type)) or 19 amino acids (STh (human type)) and having a molecular weight of approximately 2,000, and it has been known for its very low immunogenicity so far. Studies have been made to improve immunogenicity using ST as a low-immunogenicity molecular model.
Klipstein et al. (1985) (Non-Patent Document 1) created an antigen by chemically fusing STh with LTB, encapsulating the antigen in gelatin capsules, and orally administering the capsules, thereby confirming the increased antibody titers of serum IgG and small intestinal IgA. However, further improved immunogenicity has been required for practical use.
Clements (1990) (Non-Patent Document 2) induced expression of the LTB-ST fusion protein in Escherichia coli, purified the protein, and intraperitoneally administered the protein to mice, thereby confirming induction of the anti-ST antibody in serum. However, further increase in immunogenicity has been required.
Zhang et al. (2010) (Non-Patent Document 3) induced expression of a protein obtained by fusing LT holotoxin with ST in Escherichia coli, purified the protein, and intramuscularly administered the protein with an incomplete Freund's adjuvant to rabbits, thereby confirming antibody induction and induction of neutralization activity against the ST toxin. However, since the injection containing the adjuvant was administered, it has been required to further improve immunity against the antigen.
Rosales-Mendoza et al. (2011) (Non-Patent Document 4) induced expression of a fusion protein of LTB and STh in tobacco and orally administered the protein to mice, thereby confirming the increased anti-LT antibody titer. However, Rosales-Mendoza et al. did not mention the anti-ST antibody titer.
Meanwhile, the present inventors previously reported in Japanese Patent No. 5360727 (Patent Document 1) that high production of a B subunit of Escherichia coli heat-labile toxin (LTB) or a B subunit of Shiga toxin 2e (Stx2eB) in plants was achieved by making use of a linker (PG12) having a specific amino acid sequence. However, ability of the fusion protein serving as a vaccine or ability of the antigen in a state of being fused with a third antigen has been unclear.